Humanized antibody directed to the IL-2 receptor beta-chain prolongs primate cardiac allograft survival.

IL-2Rs are expressed by T cells activated in response to foreign histocompatibility Ags but not by normal cells. This difference in IL-2R expression is exploited by blockade of IL-2Rs to achieve immunosuppression. High affinity IL-2Rs involve three subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma. Murine Mik beta 1, a mAb that blocks IL-2 binding to IL-2R beta, was developed as an immunosuppressive agent. There was modest prolongation of cynomolgus cardiac allograft survival in animals treated with murine Mik beta 1 (mean survival 11.8 +/- 1.6 days compared with 8.2 +/- 0.4 days in untreated animals; p = 0.06). However, murine Mik beta 1 is ineffective in recruiting primate effector cells and is neutralized by monkey Abs directed toward the infused Ab. To circumvent these limitations, a humanized form of Mik beta 1, which is a largely human IgG1k Ab, except that murine hypervariable regions are retained, was developed. In vivo plasma survival of humanized Mik beta 1 was threefold longer than simultaneously administered murine Mik beta 1 (terminal t1/2, 104 +/- 10 h vs 37 +/- 2 h). Furthermore, humanized Mik beta 1 manifests Ab-dependent cellular cytotoxicity, an activity that is absent with the parental murine Mik beta 1. Graft survival was significantly prolonged by humanized Mik beta 1 treatment with survivals of 22, 22, 24, 27, 44, and > 300 days (p vs control < 0.01; p vs murine Mik beta 1 < 0.01). Survival was not prolonged further (p > 0.3) by the addition of humanized anti-Tac, which blocks interaction of IL-2 with IL-2R alpha subunits. There was no toxicity attributable to the use of Mik beta 1 Abs. Thus, humanized Mik beta 1 prolonged cardiac allograft survival in primates without toxicity and may be effective as an adjunct to standard immunosuppressive therapy.

Purification and characterization of human recombinant IgE-Fc fragments that bind to the human high affinity IgE receptor.

The Fc-region of immunoglobulin E (IgE) comprising C epsilon 2, C epsilon 3, and C epsilon 4 domains is sufficient for binding to the alpha chain of the high affinity IgE-Fc receptor (Fc epsilon RI alpha). In order to identify the smallest Fc fragment capable of binding to the Fc epsilon RI alpha with high affinity, various regions of the IgE-Fc molecule were expressed in COS cells and investigated for their ability to bind Fc epsilon RI alpha. The smallest fragment that showed Fc epsilon RI alpha binding activity spans amino acids 329-547 and lacks the entire C epsilon 2 domain. Two active fragments, viz. Fc epsilon(315-547) (containing Cys328 which is responsible for interchain S-S bonding) and Fc epsilon(329-547), have been overexpressed in CHO cells and purified to homogeneity. The purified proteins bind to the Fc epsilon RI alpha with high affinity, similar to native IgE. SDS-polyacrylamide gel electrophoresis analyses indicate that Fc epsilon(315-547) is an S-S-linked dimer of apparent molecular mass of 68 kDa. Fc epsilon(329-547) appears on SDS-gel as three distinct bands at approximately 32 kDa, both under reducing and nonreducing conditions. However, size exclusion chromatography and analytical ultracentrifugation studies suggest that Fc epsilon(329-547) also remains associated as a dimer. The presence of N-linked glycosylation was detected in both proteins. The deglycosylated form of Fc epsilon(315-547) was isolated after Endo F/N-glycosidase F digestion and demonstrated to have binding activity comparable to that of the mock-digested protein. These results suggest that the presence of N-linked sugars is not necessary for Fc epsilon RI alpha binding. Both proteins blocked the release of histamine from RBL cells expressing human Fc epsilon RI alpha in a dose-dependent manner. The availability of these recombinant IgE-Fc proteins will be helpful in elucidating the key epitopes essential for the binding of IgE to its high affinity receptor.

The anti-idiotypic response by cynomolgus monkeys to humanized anti-Tac is primarily directed to complementarity-determining regions H1, H2, and L3.

The anti-ld response developed by cynomolgus monkeys to the humanized anti-Tac antibody was analyzed by using 12 humanized anti-Tac variants differing in V region structure. The majority of the monkey response was directed against idiotopes composed wholly or in part of complementarity-determining regions H1, H2, and L3. There was no detectable response directed solely to five single complementarity-determining regions examined or solely to the modified human V region framework.

Reduced immunogenicity and improved pharmacokinetics of humanized anti-Tac in cynomolgus monkeys.

The anti-Tac mAb has been shown to bind to the p55 chain of the IL-2R, block IL-2 binding and inhibit T cell proliferation. A humanized form of anti-Tac (HAT) has been constructed that retains the binding properties of murine anti-Tac (MAT). These two mAb were evaluated in cynomolgus monkeys to compare relative immunogenicity and pharmacokinetic properties. Monkeys treated with HAT daily for 14 days exhibited anti-HAT antibody titers which were 5- to 10-fold lower than their MAT-treated counterparts and these antibodies developed later than in the MAT-treated monkeys. Two of four monkeys receiving a single injection of MAT developed anti-MAT antibodies, whereas none of four monkeys developed antibodies after a single treatment with HAT. In monkeys injected with either HAT or MAT daily for 14 days, the anti-antibody titers induced were inversely related to the amount of anti-Tac administered. Antibodies that developed against MAT were both anti-isotypic and anti-idiotypic, whereas those developed against HAT appeared to be predominantly anti-idiotypic. The pharmacokinetic properties, that is the half-life and area under the curve values, of HAT were also significantly different from those of MAT. The area under the curve values for HAT in naive monkeys were approximately twofold more than those for MAT, and the mean serum half-life of HAT was 214 h, approximately four- to fivefold more than MAT. These pharmacokinetic values were reduced in monkeys previously sensitized with HAT or MAT suggesting that the presence of anti-antibodies altered these parameters.

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction.

The alpha subunit of the human IgE receptor (FcERI) is sufficient for high affinity IgE binding.

The alpha subunit of the FcERI binds IgE with high affinity. Previous studies have demonstrated that alpha subunit expression requires the presence of beta and/or gamma subunits, and it is not known how these two subunits contribute to the ability of the alpha subunit to bind IgE. In this report, we describe the expression and characterization of a human chimeric alpha subunit. The data demonstrate that high affinity IgE binding does not require the presence of the beta and/or gamma subunits and that this activity is localized to the extracellular domain (residues 26-201) of the human alpha subunit. Permanent cell lines expressing the chimeric receptor were used to characterize the binding parameters of the alpha subunit. These cell lines provide a means of identifying therapeutic agents which may be effective in the treatment/management of allergic diseases.